The present invention relates to the field of pharmaceutical screening methods for novel drugs and for the ribonucleotide targets to which they bind.
A ribonucleoprotein (RNP) complex is formed between ribonucleic acid (RNA) and protein. Such RNPs are shown to participate in almost all macromolecular processes, including RNA processing, protein synthesis RNA editing, and the signal recognition of proteins targeted for export. Knowledge of the functional importance of RNA is ever increasing, as exemplified by the indication that, for example, 23S rRNA plays a key role in peptidyl transferase [H. Noller et al., Science, 256:1416-1419 (1992)]. The translation apparatus (i.e., that which decodes RNA for protein synthesis) is essential to all living cells and represents one of the major targets for antibiotics and other pharmaceutically useful compositions.
An understanding of the precise mechanism of drug action is dependent on detailed knowledge at the molecular level, of the structure and function of the drug-RNP complex, e.g., the ribosome and its associating factors. However, such understanding at the molecular level of RNA structure and RNA-ligand interactions has been hampered by the size and complexity of, for example, the ribosome and other RNP particles.
As one example of RNP particles, the ribosome is likely to have evolved from autonomously assembled structural sub-domains. Domain organization occurs within the ribosome. Partial ribonuclease digestion of the 30S subunit releases a RNP complex containing ribosomal proteins (r-proteins) S7, S9, S19, S13, or S14 and fragments derived from the 3xe2x80x2 half of 16S rRNA [J. Morgan et al., Eur. J. Biochem., 29:542-552 (1972); A. Yuki et al, Eur. J. Biochem., 56:23-34 (1975)]. Specific RNP particles encompassing the 5xe2x80x2 and central domains have also been isolated [R. A. Zimmerman, Ribosomes, (Nomura, M. et al. eds.) Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1974), p. 225-269]. The small subunit ribosomal RNA (rRNA) from E. coli (16S rRNA) is organized into three major domains: the 5xe2x80x2, central and 3xe2x80x2 domains. Ribosomal RNA fragments representing each of these subdomains can reassemble with specific subsets of ribosomal proteins [Noller, H. F. et al, Science, 212: 402-411 (1981)].
Several lines of evidence support the notion that the ribosome can be fragmented into smaller, functional subdomains that retain organizational and ligand-binding properties characteristic of the intact particle. For example, in vitro assembly of intact 30S subunits have further demonstrated two to three independent nucleation events for various domains within the 30S subunit. This provides a further indication for the existence of independent assembly domains [Nomura, M. et al, J. Cell Physiol., 74: 241-252 (1974); W. A. Held et al., J. Biol Chem., 249:3103-3111 (1974)].
More recently, a fragment corresponding to the 5xe2x80x2 domain (nucleotides 1-526) assembled with r-proteins S4, S16, S17, and S20 [C. J. Weitzmann et al., FASEB J., 7:177 (1993)], and a fragment of the 3xe2x80x2 domain of 16S rRNA (nucleotides 923-1542) reconstituted together with eight r-proteins formed a structure that resembled the head of the 30S subunit [Samaha, R. R. et al, Proc. Natl. Acad. Sci., 91:7884-7888 (1994)]. This particle retains the property of being able to bind the antibiotic, spectinomycin, which specifically protects the N-7 position of G1064 from attack by dimethylsulphate in both 30S subunits and the sub-particle [Samaha et al, cited above; Moazed, D. et al, Nature (London), 327: 389-394 (1987)].
The degree of protection to both particles shows the same dependence on drug concentration, indicating that spectinomycin binds with similar affinity to each particle. Resistance mutations have been mapped to structural changes in either ribosomal protein S5 or within helix 34 of 16S rRNA formed by base pairing between nucleotides 1046-1065 and 1191-1211 [Brink, M. F. et al, Nucleic Acids Res., 22:325-331 (1994) and Johanson, U. et al, Nucleic Acids Res., 23:464-466 (1995)]. See FIG. 1.
Further dissection of the ribosome has been achieved and expanded to include interactions with ligands other than ribosomal proteins. An oligoribonucleotide analogue of the decoding region located near to the 3xe2x80x2 end of 16S rRNA interacts with both antibiotic (neomycin) and RNA ligands (tRNA and mRNA) of the 30S subunit in a manner that resembles normal subunit function {Purohit, P. et al, Nature (London), 370: 659-662 (1994)}. Accordingly, fragmentation of RNP complexes and indeed large RNAs can be considered as a potent strategy in the analysis of such molecules [Schroeder, R., Nature (London), 370:597-598 (1994)].
The binding of the antibiotic, spectinomycin, is independent of S5 indicating that the rRNA is the major determinant of the binding site [Samaha et al, cited above]. The G1064-C1192 base pair is likely directly involved in the binding of spectinomycin as revealed by the chemical footprinting data and the existence of resistance mutations that reflect either a disruption of the base pair or replacement of the base pair [Brink et al, cited above].
Mutations in E. coli 16S rRNA that confer spectinomycin resistance include C1192U,G,A, G1064U,C,A, G1064U-C1192A, G1064-C1192G, G1064A-C1192U and C1066U. The major effect of spectinomycin in vitro is proposed to inhibit the translocation of peptidyl-tRNAs from the A-site to the P-site by preventing the binding of elongation factor G (EF-G) to the ribosome [Bilgin, N. et al., EMBO J., 9: 735-739 (1990)]. Helix 34 has been proposed to melt during the elongation cycle and spectinomycin exerts its inhibitory effect by stabilizing the helix [Brink et al, cited above]. Helix 34 has the potential to exhibit two structural conformers similar to the phylogenetic model, without disruption of the overall base pairing arrangement [Prescott, C. D. et al., Biochimie 1991, 73, 1121-1129] (See FIG. 2). The conformers reflect the alternate availability of either an xe2x80x9cupperxe2x80x9d (1199-1201) or xe2x80x9clowerxe2x80x9d(1202-1204) 5xe2x80x2-UCA-3xe2x80x2 triplet.
To date, characterization of the interaction between drug and rRNA has been based on the above-described in vitro approaches, for example, ligand binding to an oligoribonucleotide analogue of the decoding region located near to the 3xe2x80x2 end of 16S rRNA. This RNA fragment interacts with both antibiotic (neomycin) and RNA ligands (tRNA and mRNA) of the 30S subunit in a manner that resembles normal subunit function.
Despite the wealth of research in this area, there remains a need in the art for methods and compositions useful for identifying pharmaceutically useful compounds, e.g., antibiotics, which bind cellular RNA targets.
As one aspect, the present invention provides a method for identifying an RNA fragment that mimics the structure of a binding site of a target RNA molecule (hereafter referred to as a xe2x80x9cmimicking RNA fragmentxe2x80x9d). In this method, the target molecule is a defined, known RNA molecule. The method includes the steps of providing a defined DNA fragment, by either fragmenting DNA encoding the target RNA molecule with one or more restriction enzymes or chemically synthesizing a DNA fragment encoding a portion of the RNA target molecule. The defined fragment is cloned into a plasmid which, under suitable conditions, permits synthesis of the RNA fragment encoded by the DNA fragment. The plasmid is transfected into a host cell which contains the target RNA molecule. Untransfected host cells are cultured in the presence of a compound which inhibits cell growth or kills the cells. The transfected cells are similarly cultured in the presence of the compound. If the transfected cells permit the synthesis of an RNA fragment that mimics the target molecule, the RNA fragment imparts drug resistance to the transfected cells, which show no appreciable defect in growth. Thereafter, plasmid containing the DNA fragment, which encodes the mimicking RNA fragment, is isolated from the host cell and characterized by conventional means. This defined DNA fragment thus provides a defined mimicking RNA fragment.
In another aspect, the invention provides a method for screening for compounds which bind a mimicking RNA fragment as described above. The method involves providing as a control a untransfected host cell and providing a host cell transfected with a plasmid comprising a DNA sequence encoding the mimicking RNA fragment. Both cells are exposed to a library of compounds. One or more compounds is identified which, inhibits the growth of, or kills, the untransfected cells. One or more of those compounds is identified which has no effect on the transfected cells. The compound meeting both requirements is identified as binding to the defined RNA fragment contained in the transfected cells.
In yet a further aspect, the invention provides another method for identifying an RNA fragment that mimics the structure of a binding site of a defined target RNA molecule, but uses random, not defined, DNA. In this method, the random DNA fragments are provided from the DNA which encodes the defined target. This is done by randomly fragmenting the DNA encoding the target with one or more restriction enzymes to produce multiple and random DNA fragments or by chemically synthesizing random fragments. As described above, a plasmid library is prepared by cloning each fragment into an identical plasmid. The library thus contains plasmids which under suitable conditions permit synthesis of the RNA fragments encoded by the DNA fragments. Following transfection of host cells with the plasmid library, the transfected cells are cultured in the presence of a compound capable of inhibiting growth of, or killing, untransfected host cells. Cells transfected with a plasmid that results in the synthesis of an RNA fragment that mimics the target molecule, are resistant to the compound. Plasmids from the resistant cells are isolated and the DNA encoding the random mimicking RNA fragments are identified and characterized. This method permits identification of a random mimicking RNA fragment from a defined target.
In yet a further aspect of this invention, a method is provided for screening for compounds which bind the random mimicking RNA fragment by providing an untransfected host cell as a control and a host cell transfected with plasmids, each plasmid comprising a random DNA sequence encoding a random mimicking RNA fragment. Both the control and transfected cells are exposed to a library of putative binding compounds. One or more compounds are identified which meet two requirements: (1) inhibit the growth of, or destroy, the controls; and (2) permit normal cell growth in some transfected cells, thereby identifying the compound as binding to a mimicking RNA fragment. A plasmid which contains a random DNA encoding a mimicking RNA fragment is isolated from the unaffected transfected cells, and the random DNA encoding the mimicking RNA fragment is identified and characterized. This method produces a defined DNA fragment encoding a defined RNA fragment for further screening, as provided by the first method described above.
In still another aspect, the invention provides a method for identifying a mimicking RNA fragment of an undefined target RNA molecule as well as a compound that binds the RNA fragment. The method comprising the steps of:
(a) providing random DNA fragments by either randomly fragmenting DNA from a selected source with one or more restriction enzymes to produce multiple DNA fragments or by chemically synthesizing random DNA fragments;
(b) cloning each fragment into an identical plasmid resulting in a library of plasmids which under suitable conditions permits synthesis of the RNA fragments encoded by the DNA fragments, thereby generating a random RNA fragment library;
(c) transfecting the plasmid library into a host cell which contains the RNA target;
(d) culturing the transfected cells in the presence of a library of compounds;
(e) identifying cells which exhibit growth inhibition or cessation in the presence of at least one compound;
(f) identifying cells which are resistant to the deleterious growth effects in the presence of the same compound, which indicates that an RNA fragment expressed by the transfected cell confers resistance to the compound on the cell;
(e) identifying the compound;
(f) isolating plasmids from the resistant cells; and
(g) identifying and characterizing from the plasmids of step (f) the DNA encoding a mimicking RNA fragment of the undefined target.
According to one embodiment of the latter method, the method further includes comparing the secondary structure of two or more RNA fragments identified in step (g) which confer resistance to the same compound. A DNA fragment which encodes a common RNA structural motif in the two or more RNA fragments is identified by resort to RNA bioinformatics computer algorithms known in the art. This method thereby aids the identification of a defined RNA fragment of the undefined target molecule.
According to still another embodiment of the latter method, another step involves screening a library of RNA sequences with the defined RNA fragment to determine the source of the defined RNA fragment and identifying the RNA target molecule thereby.
In yet another aspect, the invention provides as a composition an RNA fragment which mimics a binding site of an intracellular RNA molecule, e.g., an RNA fragment which mimics a binding site for spectinomycin. These RNA fragments are identified by the methods described above.
In still a further aspect, the invention provides a DNA sequence which encodes the mimicking RNA fragments.
In yet a further aspect, the invention provides pharmaceutically useful compounds and compositions, which may be antibiotics, bacteriostatics, or compositions which interact with mRNA to alter expression levels of proteins, identified by the methods described above.
Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.